title: Cloning, expression, and characterization of chicken tissue inhibitor of metalloproteinase-2 (TIMP-2) in normal and transformed chicken embryo fibroblasts.
abstract: Rous sarcoma virus-transformed chicken embryo fibroblasts (RSVCEF), when compared to normal CEF, produce elevated levels of matrix metalloproteinase-2 (MMP-2) that exists in a form free of complexed tissue inhibitor of metalloproteinase-2 (TIMP-2). In order to ascertain whether the increased levels of TIMP-free MMP-2 in RSVCEF cultures are due to diminished expression of TIMP-2 or alterations in TIMP-2 that diminish its MMP-2 binding ability, it was necessary to clone, characterize, and express chicken TIMP-2 cDNA. The TIMP-2 cDNA was cloned from a chick embryo lambda gt11 library by RT-PCR using primers based on amino-acid sequences determined from isolated TIMP-2. The deduced amino acid sequence for chicken TIMP-2 is 81% identical to human TIMP-2; most of the sequence differences lie in the carboxyl terminal portion of chicken TIMP-2. Northern analysis of mRNA levels in CEF and RSVCEF demonstrates that TIMP-2 mRNA levels are increased in RSVCEF. However, TIMP-2 protein levels, relative to proMMP-2 levels, appear to decrease upon transformation and suggest additional control of TIMP-2 at the post-transcriptional level. Addition of recombinantly expressed TIMP-2 to RSVCEF cultures causes a disappearance of TIMP-free (TF) proMMP-2 with a corresponding increase in the TIMP-complexed (TC) proMMP-2 levels, demonstrating that TF proMMP-2 is capable of converting to TC pro-MMP-2 when free TIMP-2 is available. Surprisingly, RSVCEF cultures manifest a TIMP-2 population that is not complexed to MMP-2, despite the coexistence of TIMP-free proMMP-2. Gel-filtration analysis indicates that this uncomplexed TIMP-2 exhibits an apparent molecular weight of 50 kDa, indicating it is not free TIMP-2 and that it exists in transformed cultures in a noncovalent complex with an undefined molecule. Thus transformed cells can alter the TIMP-2/MMP-2 balance by transcriptional and post-translational modifications, yielding a population of inhibitor-free, proteolytically active MMP2.
authors.affiliation: Department of Biochemistry, State University of New York at Stony Brook 11794-8691, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Aimes
authors.forename: L H
authors.initials: LH
authors.lastname: Li
authors.forename: B
authors.initials: B
authors.lastname: Weaver
authors.forename: S
authors.initials: S
authors.lastname: Hawkes
authors.forename: E A
authors.initials: EA
authors.lastname: Hahn-Dantona
authors.forename: J P
authors.initials: JP
authors.lastname: Quigley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses; D001483:Base Sequence; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000235:genetics*; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D004792:Enzyme Precursors / Q000096:biosynthesis; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D018093:Gelatinases / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
chemical_list: D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D004792:Enzyme Precursors; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2